CN111373037B - Chitin-decomposing enzyme composition, chitin decomposition reaction liquid, and method for producing sugar - Google Patents
Chitin-decomposing enzyme composition, chitin decomposition reaction liquid, and method for producing sugar Download PDFInfo
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- CN111373037B CN111373037B CN201880075281.3A CN201880075281A CN111373037B CN 111373037 B CN111373037 B CN 111373037B CN 201880075281 A CN201880075281 A CN 201880075281A CN 111373037 B CN111373037 B CN 111373037B
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Abstract
The chitin-degrading enzyme composition comprises: chitinase as a chitinase for hydrolyzing chitin; silica or a silica-containing substance; and a nitrogen-containing heterocyclic compound as a nitrogen-containing heterocyclic compound.
Description
Technical Field
The present invention relates to a chitinase composition, a chitin decomposition reaction solution, and a method for producing a sugar.
Background
Chitin is contained in the outer skeletons of insects and crustaceans, cell walls of fungi, and the like in the biological world, and is presumed to be produced second to cellulose, which is biomass that is present abundantly on the earth, in an annual amount. Therefore, chitin is gaining attention as a biomass resource next to cellulose only.
Chitin is an insoluble polysaccharide in which N-acetyl-D-glucosamine, which is a monosaccharide, is bound, and is hydrolyzed into chitin having a reduced molecular weight (low-molecular-weight chitin), chitin oligosaccharide derived from chitin, a monosaccharide (N-acetyl-D-glucosamine), and the like, by an enzymatic reaction using chitinase, which is a chitinase for degrading chitin (also referred to as "chitinase"). In addition, these hydrolysates are the products of enzymatic reactions of chitin.
The product of the enzymatic reaction of chitin has excellent antibacterial property, moisture retention, biocompatibility, safety, chelating property, etc. Therefore, the use of the polymer in various fields such as medical materials, medicines, cosmetics, fibers, agriculture, water treatment, and foods is expected, and research and development are being carried out.
For example, non-patent document 1 describes a technique relating to the enzymatic activity of chitinase using Silica Nanoparticles (SNPs). Specifically, it is disclosed that a chitinase (Chi 9602) derived from Bacillus thuringiensis (Bacillus thuringiensis) is immobilized on the surface of SNP by electrostatic adsorption to prepare a nanoscale chitinase (SNPC 1), and the enzymatic activity of the chitinase is measured using the prepared SNPC 1. The immobilization rate of silica in the prepared SNPC1 was about 55%.
However, since the chitinase activity of SNPC1 was about 43% relative to the chitinase activity of Chi9602 of 100%, it was found that the activity was reduced by about 57% by immobilization of silica. That is, it was revealed that although a reaction system in which chitinase is immobilized on silica can produce sugar, the enzyme activity is inhibited to lower the efficiency of the saccharification reaction, and it is necessary to solve such a problem when using this reaction system. Further, a complicated reaction system is not desirable from the viewpoint of cost.
Documents of the prior art
Non-patent literature
Non-patent document 1: qin et al, international Journal of Biological Macromolecules, vol.82, 2016, p.13-p.21
Disclosure of Invention
Problems to be solved by the invention
The present invention has been made in view of the above circumstances, and an object thereof is to provide a chitinase composition, a chitinase solution, and a sugar production method, which can improve the efficiency of a saccharification reaction using a chitinase by a simple process and can realize a high yield of a product, when silica is applied to a reaction system using the chitinase.
Means for solving the problems
The invention to achieve the above object, according to claim 1, provides a chitin-decomposing enzyme composition comprising: chitinase as a chitinase for hydrolyzing chitin; silica or a silica-containing substance; and a nitrogen-containing heterocyclic compound as a nitrogen-containing heterocyclic compound.
The invention according to claim 2 is the chitinase composition according to claim 1, characterized in that the chitinase at least contains chitinase A.
The invention according to claim 3 is the chitinase composition according to claim 1 or 2, wherein the nitrogen-containing heterocyclic compound is a nitrogen-containing five-membered heterocyclic compound.
The 4 th aspect of the present invention is the chitin-degrading enzyme composition according to the 3 rd aspect, wherein the nitrogen-containing five-membered heterocyclic compound is 1 selected from the group consisting of imidazole and 2-methylimidazole.
The invention according to claim 5 for achieving the above object is a chitin decomposition reaction liquid comprising: chitin; and any one of the chitinase compositions of claims 1 to 4.
The present invention according to claim 6 for achieving the above object is a method for producing a saccharide, characterized in that a saccharide is produced by hydrolyzing chitin using the chitin decomposition reaction liquid according to claim 5.
The 7 th aspect of the present invention is the method for producing a saccharide according to the 6 th aspect, wherein the saccharide is produced by hydrolyzing chitin using the chitin decomposition reaction liquid under stirring.
ADVANTAGEOUS EFFECTS OF INVENTION
According to the present invention, there can be provided a chitinase composition, a chitinase reaction solution, and a sugar production method, which can improve the efficiency of a saccharification reaction using a chitinase by a simple process and can realize a high yield of a product, when silica is applied to a reaction system using the chitinase.
Detailed Description
(chitin-decomposing enzyme composition)
The chitinase composition of the present invention improves the efficiency of the saccharification reaction in an enzymatic reaction using chitinase as a chitinase, and realizes high yields of oligosaccharides, N-acetyl-D-glucosamine, and the like as products. The chitin-degrading enzyme composition of the present invention will be described in detail below.
The chitin-degrading enzyme composition of the present invention is a composition capable of hydrolyzing chitin as a substrate, and contains: a chitin-degrading enzyme; silica or a silica-containing substance; and nitrogen-containing heterocyclic compounds.
Here, chitin as a substrate is a linear high molecular amino sugar in which a plurality of N-acetyl-D-glucosamine residues are bound by β -1,4 bonds, and is an insoluble polysaccharide having a strong crystal structure. Chitin has a chemical structure represented by the following formula (1).
The deacetylated product of chitin is called Chitosan (Chitosan) and has a chemical structure represented by the following formula (2).
General chitin has a chitosan structure partially deprived of acetyl groups, and on the other hand, chitosan has a chitosan structure partially having acetyl groups. Therefore, chitin in the present invention is a concept including chitin having a chitosan structure as described above as well as chitosan having a chitosan structure. Further, such chitin may be a chitin-containing substance containing chitin to the extent that the product in the enzymatic reaction can be recovered.
The raw material of the chitin is not particularly limited, and a raw material derived from chitin-based biomass can be used. Examples of such a raw material include shells of shrimps, crabs, etc., soft shells of cuttlefish, etc., shells or exoskeletons of insects, and cell walls of fungi. In addition, natural products or commercially available products may be used as the raw materials. When the raw materials are derived from natural sources, 1 kind of them may be used alone, or 2 or more kinds may be used in combination.
As a raw material derived from a natural product, for example, red crab can be used. In the case of red snow crab, the chitin can be obtained by crushing the dried red snow crab shell, the leg from which the trunk is removed, and the joint part thereof into 3-5 cm, immersing them in a high-temperature dilute aqueous sodium hydroxide solution and a room-temperature dilute aqueous hydrochloric acid solution for 2-3 hours, respectively, and removing the protein and calcium carbonate. Chitosan can be obtained by deacetylating the obtained chitin in a high-temperature concentrated aqueous sodium hydroxide solution for 8 to 20 hours. When the time required for deacetylation is short, the chitosan obtained becomes a chitosan having a chitin structure, which is a substance having a low degree of deacetylation. The chitin obtained in this way, or chitosan having a chitin structure as required, can be subjected to an enzymatic reaction using the chitin-degrading enzyme composition of the present invention by the steps described later.
Further, it is known that 2 crystal structures of α -chitin and β -chitin exist in chitin existing in nature.
The chitinase composition of the present invention is such that either one of the structures of alpha-chitin and beta-chitin can be hydrolyzed, or chitin containing these structures can be mixed and hydrolyzed. It is also considered that chitosan has both α and β structures, as in chitin, and chitosan having either structure can be hydrolyzed as long as it has a chitin structure.
In the present invention, chitinase is mainly used as the chitinase. Such chitinase is a product such as chitin having a low molecular weight (low molecular weight chitin), chitin oligosaccharide derived from chitin, monosaccharide (N-acetyl-D-glucosamine), or the like, which is obtained by hydrolyzing chitin and acts as an enzyme in an enzymatic reaction.
The source of the chitinase is not particularly limited, and may be, for example, a microbial source, a plant source, an insect source, or the like, and among them, chitinase derived from a microorganism is preferable. The microorganism producing chitinase is not particularly limited, and examples thereof include bacteria of the genus Serratia (Serratia), genus Bacillus (Bacillus), genus Streptomyces (Streptomyces), genus Alteromonas (Alteromonas), genus Coccidioides (Coccidioides), genus Vibrio (Vibrio), and the like; fungi belonging to the genera Aspergillus (Aspergillus), trichoderma (Trichoderma), and the like.
Further, the plant producing chitinase is not particularly limited, and examples thereof include pararubber tree (Hevea brisiliennsis), soybean (Glycine max), tobacco (Nicotiana tabacum), and the like.
Further, the insect producing chitinase is not particularly limited, and examples thereof include silkworm (Bombyx mori), spodoptera litura (Spodoptera litura), and the like.
Among them, preferred are chitinases derived from Serratia marcescens (Serratia marcescens), streptomyces coelicolor (Streptomyces coelicolor), streptomyces griseus (Streptomyces griseus), and Bacillus thuringiensis (Bacillus thuringiensis), respectively.
In addition, these chitinases may be artificially altered (e.g., cloning in the examples described below). These chitinases may be used alone in 1 kind, or may be used in combination in 2 or more kinds. In addition, chitinases may be a series of enzyme groups. Examples of such an enzyme group include chitinase (EC 3.2.1.14) and the like. In addition, chitinases of different origins may be used in combination.
It is known that chitinases have a plurality of steric structures, but the steric structure is not particularly limited when used as a chitinase, and 1 type may be used alone or 2 or more types may be used in combination. Examples of chitinases having such a three-dimensional structure include chitinase A or B derived from Serratia marcescens, chitinase A1 derived from Bacillus circulans WL-12, chitinase derived from pathogenic filamentous fungi Coccidioides immitis, ヘバミン derived from Palakegum, chitinase derived from Pyrococcus furiosus, chitinase C derived from Streptomyces actinomyces griseus, and the like.
Among them, chitinase a is preferable, and one containing at least chitinase a is particularly preferable. In addition, chitinases derived from microorganisms and plants have a mixed structure. In this case, the chitinase may be purified by a separation method such as size exclusion chromatography (size exclusion chromatography). The separation of the purified chitinase into each structure can be achieved by, for example, changing the timing of sampling by size exclusion chromatography according to the molecular weight of the chitinase.
In general, chitinases are mostly enzymes having an optimum enzymatic activity in the range of pH3 to pH8, but may be enzymes called alkaline chitinases having an optimum enzymatic activity in the range of pH8 to pH10 or more. In addition, most chitinases have the most suitable enzyme activity at a reaction temperature in the range of 25 ℃ to 50 ℃, but may be an enzyme called thermostable chitinase having the most suitable enzyme activity at a reaction temperature in the range of 70 ℃ to 100 ℃.
In the present invention, as the silica or the silica-containing substance, silica, diatomaceous earth, silica sand, quartz, glass, or the like can be used. Among the substances containing silica, diatomaceous earth and silica sand are natural substances containing silica as a main component. Silica is a generic term for compounds containing at least silica, and silanol groups are generally present on a part of the surface. The silica particles may be spherical or non-spherical, may have a solid or porous particle structure, may have amorphous or crystalline crystallinity, and may be used in any form of powder, suspension, dispersion, or the like. A portion of the silica surface may be modified with other functional groups than silanol groups. Further, the layer may be in the form of a layer in which silicon dioxide is present by reacting the surface of a compound other than silicon dioxide with a silane coupling agent, a silicon alkoxide, a silicic acid ion, or the like. Among them, colloidal silica, diatomaceous earth and silica sand are particularly preferably used.
The colloidal silica has an average primary particle diameter of 1nm to 400nm, preferably 5nm to 350nm, and is used in the presence of a chitin decomposition reaction solution described later. The average primary particle diameter is determined from the specific surface area S (m) measured by the nitrogen adsorption method (BET method) 2 The ratio of (g)/g) is calculated by the formula (D (nm) = 2720/S). Colloidal silica is used as a dispersion liquid dispersed in a dispersion medium such as water, methanol, ethanol, acetone, methyl ethyl ketone, or ethylene glycol, and the dispersion liquid is called a colloidal liquid, a sol, or the like. In the present invention, the dispersion medium may be selected within a range in which the activity of the enzyme is not inhibited, but it is preferable to use a dispersion medium such as water or ethanol.
As a method for producing colloidal silica, there are a water glass method using water glass as a raw material, an alkoxide method using a metal alkoxide as a raw material, a gas phase method using a silicon chloride compound as a raw material, and the like. Colloidal silica obtained by any manufacturing method may be used, but it is preferable to use colloidal silica obtained by a water glass method.
In the present invention, the nitrogen-containing heterocyclic compound is a nitrogen-containing heterocyclic compound. Examples of such nitrogen-containing heterocyclic compounds include a tricyclic heterocyclic compound (nitrogen-containing tricyclic heterocyclic compound) such as aziridine-methyl 2-carboxylate, 1- (2-hydroxyethyl) aziridine, and the like; five-membered heterocyclic compounds (nitrogen-containing five-membered heterocyclic compounds) such as pyrrolidine, 1H-pyrrole, 2H-pyrrole, 3H-pyrrole, imidazole, L-histidine, 2-methylimidazole, 2-ethylimidazole, benzimidazole, pyrazole, 3,5-dimethylpyrazole, 1H-1,2,3-triazole, 1,2,4-triazole, and 1H-tetrazole; six-membered heterocyclic compounds (nitrogen-containing six-membered heterocyclic compounds) such as piperidine, 2-methylpiperidine, 4-hydroxy-1-methylpiperidine, pyridine, 2-aminopyridine and 2-amino-6-methylpyridine; seven-membered heterocyclic compounds (nitrogen-containing seven-membered heterocyclic compounds) such as epsilon-caprolactam, epsilon-thiocaprolactam, 1,8-diazabicyclo [5.4.0] -7-undecene, and the like.
Among them, preferred are pyrrolidine, imidazole, L-histidine, 2-methylimidazole, pyrazole, 3,5-dimethylpyrazole, 1,2,4-triazole, 2-methylpiperidine, 4-methylpiperidine, 2-aminopyridine, 2-amino-6-methylpyridine, 1,8-diazabicyclo [5.4.0] -7-undecene, and particularly preferred are imidazole and 2-methylimidazole, from the viewpoint of being inexpensive and readily available.
(method for producing chitin decomposition reaction solution and sugar)
In the present invention, a chitin decomposition reaction solution is prepared and used in hydrolyzing chitin. The chitin decomposition reaction liquid of the present invention contains chitin as a raw material and the chitin-decomposing enzyme composition of the present invention. As will be described in detail later, in the chitin decomposition reaction liquid of the present invention, silica or a silica-containing substance and a nitrogen-containing heterocyclic compound are used in combination from the viewpoint of enjoying an effect of improving the efficiency of the saccharification reaction.
In general, in an enzymatic reaction, a phenomenon in which the reaction rate is saturated is observed when the substrate concentration is high, and the reaction rate draws a hyperbolic curve that reaches the maximum rate of saturation. This is because enzyme molecules are often larger than substrates and the range of active centers is narrow, and therefore, it is considered that the frequency of reaction between a substrate and a catalyst (enzyme) is smaller than that of a metal catalyst or the like even when the substrate and the catalyst collide with each other (are suitable for active centers). Moreover, if the substrate concentration is increased, the substrates compete with each other for fewer active centers of the enzyme, and thus a saturation phenomenon occurs.
Therefore, in the chitin decomposition reaction liquid of the present invention, the concentration of the chitin-decomposing enzyme may be determined appropriately according to the content of chitin. However, if the concentration of the chitinase in the reaction solution is too low, the efficiency of the saccharification reaction of the chitinase is lowered, which is not preferable. On the other hand, if the concentration of the chitinase is too high, not only the saturation phenomenon occurs, but also the chitinase is difficult to dissolve in the chitin decomposition reaction solution, which is economically undesirable.
In the chitin decomposition reaction liquid of the present invention, the concentration of silica in the silica or silica-containing substance is 0.1 mg/mL-400 mg/mL, preferably 0.5 mg/mL-100 mg/mL. If the concentration of silica in the silica or the silica-containing substance is less than 0.1mg/mL, the efficiency of the saccharification reaction by the chitin-degrading enzyme is lowered, which is not preferable. On the other hand, if the concentration of these silicas is higher than 400mg/mL, not only the dispersibility of the chitin decomposition reaction liquid is deteriorated, but also it is economically unfavorable.
In the chitin decomposition reaction liquid, the mass ratio of chitin-decomposing enzyme to silica or silica-containing substance (chitin-decomposing enzyme/silica) is 0.0002 to 300, preferably 0.002 to 30. If the mass ratio of the two exceeds the above range, the efficiency of the saccharification reaction by the chitinase is not significantly improved.
The concentration of the nitrogen-containing heterocyclic compound in the reaction liquid for decomposing chitin is 0.005 mg/mL-100 mg/mL, preferably 0.05 mg/mL-50 mg/mL. More preferably 0.68mg/mL to 34mg/mL. Most preferably 6.8mg/mL to 34mg/mL. If the concentration of the nitrogen-containing heterocyclic compound is less than 0.005mg/mL, the efficiency of the saccharification reaction by the chitin-degrading enzyme is lowered, which is not preferable, and if it is more than 100mg/mL, the dispersibility of the chitin-degrading reaction solution is deteriorated, which is not economically preferable.
In the chitin decomposition reaction liquid, the mass ratio of silica to nitrogen-containing heterocyclic compound (nitrogen-containing heterocyclic compound/silica) in the silica or silica-containing substance is 0.0001 to 100, preferably 0.001 to 10. If the mass ratio of the both exceeds the above range, the improvement of the saccharification reaction efficiency of the chitin-degrading enzyme is not significant.
The pH of the chitin decomposition reaction solution is 4 to 8, preferably 5 to 7. If the pH is less than 4, the silica or silica-containing substance aggregates to lower the efficiency of the saccharification reaction by the chitinase, and if the pH is more than 8, the silica or silica-containing substance is liable to dissolve in the chitin decomposition reaction solution, which is not preferable. In addition, in the case where alkaline chitinase is used in the chitin decomposition reaction liquid, the solubility of silica or a silica-containing substance in the chitin decomposition reaction liquid is adjusted. Thus, the reaction solution can be used as a chitin decomposition reaction solution having a pH of more than 8.
Examples of the pH adjuster of the chitin decomposition reaction solution include inorganic acids such as sulfuric acid, hydrochloric acid, and nitric acid; carboxylic acids such as acetic acid and oxalic acid; hydroxy acids such as citric acid, tartaric acid, malic acid, etc.; phosphoric acid; hydroxide salts such as sodium hydroxide and potassium hydroxide; and nitrogen-containing compounds such as ammonia and urea. The type and concentration of the surfactant are not particularly limited as long as the effect of the present invention is not impaired. These pH regulators may be used alone in 1 kind, or in a mixture of 2 or more kinds, or may be used in the form of a buffer solution having a buffering action.
The reaction temperature of the chitin-degrading reaction solution is preferably set in accordance with the optimum temperature of the chitin-degrading enzyme, and is, for example, preferably 10 to 50 ℃ and particularly preferably 25 to 40 ℃ or lower. In general, if the reaction temperature is lower than 10 ℃ the saccharification reaction efficiency of the chitinase is significantly reduced, and if it is higher than 50 ℃ the chitinase may be inactivated and thus is not preferred. However, when a thermostable chitinase is used, it is not inactivated even at 70 to 100 ℃.
The pretreatment of the raw material derived from chitin-based biomass may be carried out by applying a known treatment method. For example, after physical pulverization by a chopper or the like, protein and calcium carbonate may be removed by acid treatment and/or alkali treatment to obtain a material of chitin.
The procedure for preparing the chitin decomposition reaction liquid is not particularly limited, and silica or a silica-containing substance and a nitrogen-containing heterocyclic compound may be added to a dispersion in which a chitin-decomposing enzyme is dispersed to prepare the chitin decomposition reaction liquid. Alternatively, a chitin degradation reaction solution may be prepared by adding a chitin degrading enzyme to a dispersion in which silica or a silica-containing substance and a nitrogen-containing heterocyclic compound are dispersed. In these production steps, the order of addition may be any if the saccharification reaction efficiency of the chitosanase is not decreased. For example, the silica or silica-containing substance and the nitrogen-containing heterocyclic compound may be added simultaneously or separately. In this case, the nitrogen-containing heterocyclic compound may be added in the form of powder or may be added in the form of solution. The other additives such as a pH adjuster may be added in any order as long as the effects of the present invention are not impaired.
As described above, when a chitin decomposition reaction liquid is prepared using the chitin-degrading enzyme composition of the present invention, although the mechanism is not clear, by using silica or a silica-containing substance in combination with a nitrogen-containing heterocyclic compound, the hydrolysis of chitin can be further promoted and the efficiency of the saccharification reaction using chitin-degrading enzyme can be improved. Further, by increasing the efficiency of the saccharification reaction by the chitinase, the yield of the sugar as the product can be increased and the yield can be increased. In addition, the chitin decomposition reaction liquid can reduce the usage amount of chitin decomposition enzyme by the combined use of silicon dioxide or silicon dioxide-containing substance and nitrogen-containing heterocyclic compound, so the chitin decomposition reaction liquid is a simple reaction system and has excellent cost performance.
The saccharide can be produced by hydrolyzing chitin using a chitin decomposition reaction solution prepared using the chitin-decomposing enzyme composition of the present invention. In the production of the saccharide, chitin may be hydrolyzed using the chitin decomposition reaction solution under stirring. Specific methods for producing sugars are described in the examples below.
Examples
The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.
(1. Embodiment 1)
(1-1. Preparation of chitinase A)
Chitinase A used as a chitinase was prepared as follows.
Chitinase A (hereinafter referred to as "SmChiA") cloned from Serratia marcescens (see T.Watanabe et al, journal of Bacteriol, vol.179, no.22, 1997, p.7111-p.7117) was cloned into a plasmid vector pET27b (manufactured by ノバジェン) for constructing a large-scale protein expression system using a T7 promoter. In cloning, smChiA was constructed so that the carboxyl terminal side of SmChiA was attached with 6 repeated histidine tags.
Mass expression using Escherichia coli (E.coli) of SmChiA as a host was carried out by the following procedure.
Coli BL21 (DE 3) holding the above plasmid vector was cultured overnight at 37 ℃ with shaking in LB medium to which 50. Mu.g/mL of kanamycin was added, to obtain a culture solution. The next day, this culture solution was used as an inoculum, inoculated into 50. Mu.g/mL kanamycin オーバーナイトエクスプレス (Overnithtexpress (registered trademark)) LB medium (ノバジェン Co., ltd.), and shake-cultured overnight at 30 ℃. Next day, pellets of bacteria were produced by collecting bacteria using a centrifugal separator, and the pellets were suspended in バグバスター (Bugbuster) (manufactured by ノバジェン) to which ベンゾナーゼ (Benzonase (registered trademark)) was added (manufactured by ノバジェン). Subsequently, the suspension was left at room temperature for 30 minutes, and centrifuged again to recover the supernatant fraction.
Next, in order to purify/recover SmChiA from the above-mentioned supernatant fraction, the supernatant fraction was added to a 5X 5mL HisTrap HP column (manufactured by GE ヘルスケア). Then, the HP column was washed with a washing buffer. The washing buffer was prepared by mixing 20mM sodium phosphate buffer (pH 7.4), 0.5M sodium chloride, and 25mM imidazole.
Next, imidazole was added to the column to elute smcia while forming a concentration gradient so that the concentration became 25mM to 250mM (final concentration), and the smcia eluted fraction was recovered. The SmChiA-eluted fraction was subjected to centrifugal dialysis using ビバスピン (Vivaspin (registered trademark)) 20, and the washing buffer was replaced with 50mM sodium phosphate buffer (pH 6.0) to obtain a purified enzyme (SmChiA). The purified enzyme thus obtained was quantified by measuring the absorbance at 280 nm. Further, the molar absorption coefficient of SmChiA was calculated from The predicted amino acid sequence using "The Protocols Handbook" (see J.M. Walker, humana Press,2005, p.571-p.607).
(1-2. Measurement of chitinase Activity of chitin-decomposing enzyme composition)
In addition to smcia as the purified enzyme obtained by purification in (1-1.) above, a chitinase composition was prepared using silica as silica or a silica-containing substance and imidazole as a nitrogen-containing heterocyclic compound, and the enzyme activity (chitinase activity) was measured. The chitinase activity of the reaction system was measured by the following procedure. The composition of the prepared chitinase composition is shown in table 1 below.
First, a glass vial was charged with a rotor, and crystalline chitin (manufactured by Takara Shuzo Co., ltd.) at a final concentration of 12.5mg/mL, a 20mM sodium phosphate buffer solution (pH 6.0), smChiA at a final concentration of 0.125mg/mL, imidazole at a final concentration of 100mM, and a silica sol (manufactured by Nissan chemical Co., ltd., product name: ST-OL, acid sol) at a final concentration of 50mg/mL were added thereto to obtain a chitin decomposition reaction solution. The glass vial containing the chitin decomposition reaction mixture was placed in a vial Stirrer (Vaial Stirrer) HS-10VA (アズワン Co., ltd.), and stirred at 25 ℃ for 22 hours under conditions of maximum output. Immediately after the reaction was completed, the chitinase activity was measured by the following procedure.
The chitinase activity was measured by determining the amount of reducing sugars of chitosan oligosaccharide and N-acetyl-D-glucosamine as products by the PHBAH (p-Hydroxybenzoic acid hydrazine) method (see M.Lever, analytical Biochemistry, vol.47, no.1, 1972, p.273-p.279). Specifically, the chitin decomposition reaction solution was centrifuged (4 ℃, maximum centrifugal acceleration 15780 Xg, 5 minutes), and the supernatant was collected. Next, a PHBAH solution 2 times the amount of the collected supernatant and 2M sodium hydroxide equivalent to the amount of the supernatant were added to the collected supernatant, and the mixture was sufficiently suspended to obtain a suspension. Further, the PHBAH solution was obtained by mixing 0.1M PHBAH, 0.2M potassium sodium tartrate, and 0.5M sodium hydroxide.
The resulting suspension was reacted at 98 ℃ for 10 minutes and then quenched to 2 ℃ for 10 minutes. Then, the OD value (OD: optical Density; optical Density, optical Density) at a wavelength of 405nm was measured, and the difference in absorbance from the blank was determined using the OD value. The standard curve was prepared using N-acetyl-D-glucosamine, and the amount of reducing sugar was determined from the increase in absorbance in the supernatant, and is shown in table 1 below. The amount of reducing sugar shown in table 1 below is defined as the amount of reducing sugar when n = 3.
(2. Examples 2 to 4)
In examples 2 to 4, chitin-degrading enzyme compositions were prepared and their chitinase activities were measured in the same manner as in example 1 except that silica sol (product of Nissan chemical Co., ltd.; product name: ST-OZL-35, acid sol), silica sol (product of Nissan chemical Co., ltd.; product name: MP-4540M, na stabilized alkaline sol), and fumed silica (product of エボニック, product name: AEROSIL (registered trademark) OX50, hydrophilic fumed silica) were used as silica or silica-containing substance, respectively. The final concentration of each silica was 50mg/mL as in example 1. The composition of each chitinase composition and the amount of each reducing sugar determined are shown in table 1 below in the same manner as in example 1. The amount of reducing sugar shown in table 1 below is defined as the range of the amount of reducing sugar when n = 3.
(3. Example 5)
A chitinase composition was prepared and its chitinase activity was measured in the same manner as in example 2, except that sodium chloride was further added to the solution at a final concentration of 100mM in example 5. The composition of the chitinase composition and the amount of reducing sugar determined are shown in table 1 below in the same manner as in example 1. The amount of reducing sugar shown in table 1 below is defined as the amount of reducing sugar when n = 3.
(4. Example 6)
A chitinase composition was prepared in the same manner as in example 2 except that the final concentration of imidazole in example 6 was changed to 500mM, and the chitinase activity thereof was measured. The composition of the chitinase composition and the amount of reducing sugar determined are shown in table 1 below in the same manner as in example 1. The amount of reducing sugar shown in table 1 below is defined as the amount of reducing sugar when n = 3.
(5. Example 7)
A chitinase composition was prepared and its chitinase activity was measured in the same manner as in example 2 except that imidazole as a nitrogen-containing heterocyclic compound was changed to 2-methylimidazole in example 7. The composition of the chitinase composition and the amount of reducing sugar determined are shown in table 1 below in the same manner as in example 1. The amount of reducing sugar shown in table 1 below is defined as the amount of reducing sugar when n = 3.
(6. Comparative examples 1 to 10)
A chitinase composition was prepared and its activity was measured in the same manner as in example 5 except that in comparative example 1, silica, imidazole and sodium chloride were not added.
A chitinase composition was prepared and its chitinase activity was measured in the same manner as in example 5 except that in comparative example 2, silica and sodium chloride were not added.
A chitinase composition was prepared and its chitinase activity was measured in the same manner as in example 5, except that no silica was added to comparative example 3.
A chitinase composition was prepared and its chitinase activity was measured in the same manner as in example 5 except that in comparative example 4, silica and imidazole were not added.
Chitin lyase compositions were prepared and the chitinase activity was measured in the same manner as in examples 1 to 4, except that imidazole was not added to each of comparative examples 5 to 8.
A chitinase composition was prepared in the same manner as in example 5 except that no imidazole was added in comparative example 9, and the chitinase activity was measured.
In comparative example 10, a chitinase composition was prepared and the chitinase activity was measured in the same manner as in comparative example 3, except that the reaction was carried out by leaving the reaction mixture at 25 ℃ for 22 hours without stirring using a rotor.
In comparative examples 1 to 10, the composition of each chitinase composition and the amount of each reducing sugar determined are shown in table 1 below in the same manner as in example 1. The amount of reducing sugar shown in table 1 below is defined as the range of the amount of reducing sugar when n = 3.
(7. Evaluation of chitinase Activity)
(7-1. Chitin enzyme activity-improving Effect by addition of imidazole or sodium chloride)
The effect of improving the chitinase activity by adding imidazole or sodium chloride was examined by comparing the chitinase activity of comparative example 1 with that of comparative examples 2 to 4.
When the amounts of reducing sugars in comparative example 1 and comparative examples 2 to 4 were observed, the amounts of reducing sugars in comparative examples 2 to 4 were slightly increased compared to comparative example 1. This indicates that the hydrolysis reaction of chitin proceeded smoothly and the saccharification reaction efficiency was slightly improved in comparative examples 2 to 4, and it was clearly shown that the chitin enzyme activity was slightly improved by the addition of imidazole or sodium chloride.
(7-2. Effect of improving chitinase Activity by addition of silica)
The chitinase activity of comparative example 1 and comparative examples 5 to 8 was compared, and the effect of improving the chitinase activity by the addition of silica was examined.
When the amounts of reducing sugars of comparative example 1 and comparative examples 5 to 8 were observed, the amounts of reducing sugars were all reduced, although some differences were observed in comparative examples 5 to 8 depending on the form of silica, compared to comparative example 1. This indicates that the hydrolysis reaction of chitin did not proceed smoothly in comparative examples 5 to 8, and it was clarified that the addition of silica inhibited the chitinase activity. Therefore, it was evaluated that comparative examples 5 to 8 were inhibited from the chitinase activity of comparative example 1, and the chitinase activity-improving effect was shown as "inhibition" in table 1 below.
Since the reason why silica acts as a factor for inhibiting chitinase activity is consistent with the result shown in fig. 5 (SNPC 1) of non-patent document 1, it is considered that chitinase activity is inhibited by physically adsorbing chitinase to be immobilized on the silica surface in the state where chitinase and silica coexist.
(7-3. Effect of improving chitinase Activity by the combination of silica and imidazole)
According to the results of the above (7-1.) and (7-2.), the effects of improving the chitinase activity by the combined use of silica and imidazole were investigated by comparing the chitinase activities of examples 1 to 4 and 6 with those of comparative examples 2 and 5 to 8.
When the amounts of reducing sugars in examples 1 to 4 and comparative examples 5 to 8 were observed, the amounts of reducing sugars were increased in all of examples 1 to 4, although some differences were observed in the forms of silica with respect to comparative examples 5 to 8. Further, if the amounts of reducing sugars of examples 1 to 4 and 6 and comparative example 2 were observed, the amounts of reducing sugars of examples 1 to 4 and 6 were increased relative to comparative example 2. The reason is not clear, but it is clear that the hydrolysis reaction of chitin smoothly proceeds and the saccharification reaction efficiency is improved in examples 1 to 4 and 6, and that the chitinase activity is improved by the combined use of silica and imidazole. Therefore, it was evaluated that examples 1 to 4 and 6 had improved chitinase activities relative to comparative examples 5 to 8, and the chitinase activity-improving effect is shown as "yes" in table 1 below.
(7-4. Improving Effect of chitinase Activity by the Combined use of silica and sodium chloride)
According to the results (7-1.) to (7-3.), the effect of improving the chitinase activity by the combined use of silica and sodium chloride was investigated by comparing the chitinase activities of examples 2 and 5 with those of comparative examples 3 and 9.
When the amounts of the reducing sugars of examples 2 and 5 were observed, the amounts of the reducing sugars of both were hardly changed. Next, if the amount of reducing sugar of example 5 and comparative example 3 is observed, the amount of reducing sugar of example 5 increases relative to comparative example 3. Next, if the amounts of reducing sugars of example 5 and comparative example 9 were observed, the amount of reducing sugar of example 5 increased relative to comparative example 9. Thus, it was clarified that the reason why the chitinase activity was improved in example 5 was the combination of silica and imidazole, not the combination of silica and sodium chloride. Therefore, it is evaluated here that the chitinase activity of example 5 is improved over that of comparative example 9, and the chitinase activity improving effect is shown as "yes" in table 1 below.
(7-5. Improving Effect of chitinase Activity by combination of silica and 2-methylimidazole)
Based on the results (7-1.) to (7-4.) described above, the effect of improving the chitinase activity by the combined use of silica and 2-methylimidazole was investigated by comparing the chitinase activities of example 7 and comparative examples 2 and 6.
When the amounts of the reducing sugars of examples 2 and 7 and comparative examples 2 and 6 were observed, the amounts of the reducing sugars of examples 2 and 7 were increased compared to those of comparative examples 2 and 6. This shows that, in example 7 using 2-methylimidazole, the hydrolysis reaction of chitin smoothly proceeded and the saccharification reaction efficiency was improved, and it was confirmed that the chitinase activity was improved by using silica and 2-methylimidazole in combination, as in example 2 using imidazole. Therefore, it is evaluated here that the chitinase activity of example 7 is improved over that of comparative example 6, and the chitinase activity improving effect is shown as "yes" in table 1 below. From these results, it is also suggested that the chitinase activity may be improved by using a combination of a nitrogen-containing heterocyclic compound other than imidazole or 2-methylimidazole and silica.
(7-6. Chitin enzyme activity-improving Effect by the Presence or absence of agitation)
From the results (7-1.) to (7-5.) described above, the effect of improving the chitinase activity by the presence or absence of stirring was examined by comparing the chitinase activities of comparative example 3 and comparative example 10.
When the amounts of reducing sugars of comparative example 3 and comparative example 10 were observed, the amount of reducing sugar of comparative example 10 decreased compared to comparative example 3, and the chitinase activity decreased. The change in the enzymatic activity by the presence or absence of agitation is a phenomenon that may occur in a general enzymatic reaction system. Here, the results of evaluation that comparative example 10 inhibited the chitinase activity relative to comparative example 3, and the chitinase activity-improving effect was expressed as "inhibition" in table 1 below.
TABLE 1
The type A, B of the nitrogen-containing heterocyclic compounds in table 1 is as follows.
A: imidazole (molecular weight 68.08)
B: 2-methylimidazole (molecular weight 82.11)
From the above examples and comparative examples, it was found that when a chitin decomposition reaction solution is prepared using a chitin-degrading enzyme composition, the efficiency of the saccharification reaction by chitinase a is improved by using silica in combination with imidazole or 2-methylimidazole, and the yield of sugars as a product is improved, thereby achieving a high yield.
Industrial applicability
The present invention can be used in industrial fields where technologies including decomposition of chitin-based biomass including chitin contained in crustaceans such as crabs and shrimps, insects, fungi, and the like into low-molecular chitin, chitin oligosaccharide derived from chitin, monosaccharide, and the like are applied, for example, medical materials, medicines, cosmetics, fibers, agriculture, water treatment, foods, and the like.
Claims (5)
1. A chitin-degrading enzyme composition comprising: chitinase as a chitinase for hydrolyzing chitin; silica or a silica-containing substance; and a nitrogen-containing heterocyclic compound as a nitrogen-containing heterocyclic compound,
the nitrogen-containing heterocyclic compound is any 1 selected from imidazole and 2-methylimidazole as a nitrogen-containing five-membered heterocyclic compound.
2. The chitinase composition according to claim 1, wherein said chitinase comprises at least chitinase A.
3. A decomposition reaction liquid for chitin, comprising: chitin; and the chitin-degrading enzyme composition of claim 1 or 2.
4. A process for producing a saccharide, which comprises hydrolyzing chitin using the chitin decomposition reaction liquid according to claim 3.
5. The method for producing a saccharide according to claim 4, wherein the saccharide is produced by hydrolyzing chitin using the chitin decomposition reaction solution under stirring.
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| JP2017-225137 | 2017-11-22 | ||
| PCT/JP2018/041084 WO2019102838A1 (en) | 2017-11-22 | 2018-11-06 | Chitin-degrading enzyme composition, chitin degradation reaction solution and method for producing saccharide |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997001629A1 (en) * | 1995-06-28 | 1997-01-16 | Novo Nordisk A/S | A cellulase with reduced mobility |
| JPH10259347A (en) * | 1997-03-17 | 1998-09-29 | Nippon Paint Co Ltd | Antifouling coating composition prepared by using chitin/ chitosan |
| WO2013038940A1 (en) * | 2011-09-16 | 2013-03-21 | 花王株式会社 | Method for producing sugar |
| CN106574285A (en) * | 2014-08-07 | 2017-04-19 | 日产化学工业株式会社 | Saccharifying enzyme composition, saccharifying reaction solution, and sugar production method |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06113846A (en) * | 1991-03-20 | 1994-04-26 | Mercian Corp | Chitinase |
| JP2002272452A (en) | 2001-03-23 | 2002-09-24 | Kyowa Kasei Kk | Burkholderia cepacia, chitinase, method for producing chitinase and method for producing chitin oligosaccharide |
| JP4243266B2 (en) | 2005-09-01 | 2009-03-25 | 国立大学法人福井大学 | Chitinase from Paenibacillus genus and gene encoding it |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997001629A1 (en) * | 1995-06-28 | 1997-01-16 | Novo Nordisk A/S | A cellulase with reduced mobility |
| JPH10259347A (en) * | 1997-03-17 | 1998-09-29 | Nippon Paint Co Ltd | Antifouling coating composition prepared by using chitin/ chitosan |
| WO2013038940A1 (en) * | 2011-09-16 | 2013-03-21 | 花王株式会社 | Method for producing sugar |
| CN106574285A (en) * | 2014-08-07 | 2017-04-19 | 日产化学工业株式会社 | Saccharifying enzyme composition, saccharifying reaction solution, and sugar production method |
Non-Patent Citations (2)
| Title |
|---|
| Kinetics and mechanisms of depolymerization of alginate and chitosan in aqueous solution;H K Holme等;《Carbohydrate Polymers》;20081231;第73卷(第4期);第656-664页 * |
| 壳多糖酶研究的概况及最新进展;彭仁旺，管考梅，黄秀梨;《生物化学与生物物理进展》;19960425(第02期);第102-107页 * |
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| CN111373037A (en) | 2020-07-03 |
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